U.S. patent number 5,704,910 [Application Number 08/461,042] was granted by the patent office on 1998-01-06 for implantable device and use therefor.
This patent grant is currently assigned to Nephros Therapeutics, Inc.. Invention is credited to H. David Humes.
United States Patent |
5,704,910 |
Humes |
January 6, 1998 |
**Please see images for:
( Certificate of Correction ) ** |
Implantable device and use therefor
Abstract
Disclosed is an implantable device for delivering a pre-selected
molecule, for example, a hormone, into a mammal's systemic
circulation. The device comprises a blood permeable element that
can be anchored to an inner wall of an intact blood vessel. The
device also comprises a capsule that is held in place within the
blood vessel by the anchored blood permeable element. The capsule
encloses viable cells which produce and secrete the pre-selected
molecule into blood passing the capsule. The invention also
provides a minimally invasive method for percutaneously introducing
into a preselected blood vessel the device of the invention.
Inventors: |
Humes; H. David (Ann Arbor,
MI) |
Assignee: |
Nephros Therapeutics, Inc. (Ann
Arbor, MI)
|
Family
ID: |
23830999 |
Appl.
No.: |
08/461,042 |
Filed: |
June 5, 1995 |
Current U.S.
Class: |
604/502;
604/891.1 |
Current CPC
Class: |
A61F
2/022 (20130101); A61K 9/0024 (20130101); A61M
31/002 (20130101); A61F 2/0105 (20200501); A61F
2230/0067 (20130101); A61F 2230/005 (20130101); A61K
38/00 (20130101); A61F 2002/016 (20130101) |
Current International
Class: |
A61F
2/02 (20060101); A61F 2/01 (20060101); A61M
31/00 (20060101); A61M 031/00 () |
Field of
Search: |
;604/890.1,891.1,93,264,52 ;606/198,200 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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Jun 1994 |
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EP |
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34 47 202 C2 |
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Apr 1986 |
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DE |
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39 41 873 A1 |
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Jun 1991 |
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WO 89/04655 |
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WO |
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WO 90/06997 |
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WO |
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WO 92/15676 |
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WO |
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WO 93/06878 |
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Apr 1993 |
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WO |
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WO 94/15583 |
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Jul 1994 |
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WO |
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WO 95/00654 |
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Jan 1995 |
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WO |
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Other References
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Gene in Mouse Model of Renal Failure," J. Clin. Invest. 95:
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Tripathy et al. (1994) "Stable Delivery of Physiologic Levels of
Recombinant Erythropoietin to the Systemic Circulation by
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Culture of the Intact Microorgans or of Dispersed Islet Cells,"
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223-225..
|
Primary Examiner: McDermott; Corrine M.
Assistant Examiner: Blyveis; Deborah
Attorney, Agent or Firm: Testa, Hurwitz & Thibeault,
LLP
Claims
What is claimed is:
1. A method for percutaneously introducing into a blood vessel a
device for delivering a pre-selected molecule into systemic
circulation, the method comprising the steps of:
(a) anchoring a blood permeable element to an inner wall of all
intact blood vessel, the element when anchored permitting blood in
the vessel to pass therethrough;
(b) introducing at a location upstream of the dement a capsule
comprising viable calls which produce and secrete the pre-selected
molecule; and
(c) permitting the capsule, impelled by blood flow, to be captured
by the element.
2. The method of claim 1, comprising the additional step, prior to
step (a), of introducing the element into the vessel via a
catheter.
3. The method of claim 1 or 2, wherein the capsule is introduced
into the vessel by a catheter.
4. The method of claim 3, wherein the element is anchored in a
vein.
5. The method of claim 4, wherein the vein is selected from the
group consisting of an inferior vena cava, a superior vena cava, a
portal vein and a renal vein.
6. The method of claim 1, wherein the pre-selected molecule is a
hormone.
7. The method of claim 6, wherein the hormone is selected from the
group consisting of erythropoietin and insulin.
8. The method of claim 1, wherein the element comprises a plurality
of filaments anchorable to the inner wall of the blood vessel.
Description
FIELD OF THE INVENTION
The present invention relates to an implantable device for
delivering a pre-selected molecule into systemic circulation. More
particularly, this invention relates to an implantable device
containing viable cells. When the device is implanted into a blood
vessel, the cells produce and secrete the pre-selected molecule
into blood circulating past the device.
BACKGROUND OF THE INVENTION
The development of drug delivery devices for implantation into a
pre-selected locus in a mammal is ongoing. To date, a variety of
surgically implantable drug delivery devices have been developed
and are discussed below.
U.S. Pat. No. 4,378,016 describes a surgically implantable device
for delivering an active factor, for example, a hormone, to a
pre-selected site, for example, the peritoneal cavity, of a mammal.
The device comprises a fluid permeable membranous sack for
implantation within the mammal and an impermeable hollow tube
having one end connected to an opening in the sack and the other
end designed to remain outside the body of the mammal. The tube
provides an access passageway to the membranous sack, such that
after the sack has been surgically implanted into the mammal, a
cell containing envelope may be introduced into the sack via the
tube. Upon insertion of the cell containing envelope into the sack,
the cells may produce an active factor, which subsequently may
diffuse into the surrounding tissue or organ of the recipient.
U.S. Pat. No. 5,182,111 describes a surgically implantable device
for delivering an active factor to a pre-selected site, for
example, a tissue or organ, in a mammal. The device comprises a
semi-permeable membrane enclosing at least one cell type that
produces a specific active-factor and a second cell type that
produces an augmentory factor. The augmenting factor produced by
the second cell type subsequently induces the first cell type to
produce the active-factor.
U.S. Pat. No. 4,479,796 describes a surgically implantable
dispenser for infusing a pre-selected drug directly into the blood
stream. Briefly, the dispenser is surgically spliced in line with a
blood vessel. The dispenser encloses a replaceable cartridge of
cells, for example, micro-organisms, which produce and secrete the
drug into blood flowing past the cartridge.
U.S. Pat. No. 4,309,776 describes an intravascular drug delivery
device having a chamber containing transplanted cells for surgical
implantation into the wall of a blood vessel. The device comprises
a porous wall that permits a hormone produced by the transplanted
cells to diffuse out of the chamber and into the blood stream.
It is desirable, however, to produce a device that may be implanted
into a mammal by either non-surgical or only minimally invasive
surgical procedures, and that once implanted the device secretes a
pre-selected molecule directly into the vasculature. In addition,
it is desirable to produce a device which, when implanted,
administers the pre-selected molecule into the mammal over an
extended period and may be removed conveniently, if or whenever the
necessity arises. Accordingly, it is an object of the present
invention to provide an easily implantable device for delivering,
over long periods of time, a pre-selected molecule into the
systemic circulation of a mammal. It is another object to provide a
method for non-surgically implanting the device into a blood vessel
of a mammal for delivering the pre-selected molecule into systemic
circulation.
These and other objects and features of the invention will be more
clearly understood from the description, drawings, and claims which
follow.
SUMMARY OF THE INVENTION
The present invention provides an implantable device for delivery
of a pre-selected molecule into the systemic circulation of a
mammal. The device of the invention may be implanted using non- or
minimally invasive surgical procedures and, once implanted,
delivers the pre-selected molecule directly into the blood stream.
In addition, the device of the invention is adapted to produce in
situ and thereafter secrete the pre-selected molecule into the
blood stream over a prolonged period of time. Use of the present
device and method provides an easy and reproducible system for
delivering therapeutically effective amounts of a gene product, for
example, a hormone, growth factor, anti-coagulant, immunomodulator,
cytokine, or the like, directly into the blood stream of the
recipient. The devices of the present invention, although having a
variety of utilities, are particularly suited for use in hormone
replacement therapy.
In its broadest aspect, the device comprises a blood permeable
element, which is adapted for anchorage to an inner surface of a
blood vessel. The blood permeable element, as disclosed herein, is
designed such that when anchored to the inner surface of the blood
vessel, the element permits blood in the vessel to pass
therethrough. The device further comprises a capsule that may be
positioned, and retained in place, by contacting the element
disposed within the vessel. The capsule contains viable cells which
produce and secrete the pre-selected molecule into the blood
stream.
The term "systemic circulation" as used herein is understood to
embrace any blood vessel within a mammal, i.e., an artery,
arteriole, venule or vein, that provides a blood supply to all
tissues, except lung tissues perfused by the pulmonary circulation,
of a mammal. The systemic circulation is also referred to in the
art as the greater circulation or the peripheral circulation.
The term "blood permeable element" as used herein is understood to
mean any structure for insertion into the lumen of a blood vessel
in the systemic circulation that, once inserted, may be anchored,
for example, by hooks or barbs, to an inner surface of the blood
vessel. The element being further characterized in that when
anchored to the inner wall of the blood vessel, the element does
not occlude or prevent blood flow through the blood vessel.
In a preferred embodiment, the blood permeable element is an
embolism anti-migration filter, such as a blood clot anti-migration
filter. A variety of blood clot anti-migration filters useful in
the practice of the invention are known in the art. The currently
preferred blood permeable element is an anti-migration filter known
as a "Greenfield.RTM. vena cava filter". Useful Greenfield.RTM.
vena cava filters are described in detail in U.S. Pat. Nos.
4,817,600 and 5,059,205, the disclosures of which are incorporated
by reference herein.
The term "capsule" as used herein is understood to mean any hollow
structure dimensioned to fit within the lumen of a blood vessel,
which, when introduced into the blood vessel, does not occlude or
prevent blood flow through the vessel. The capsule is held in place
within the blood vessel by means of the blood permeable element.
For example, the capsule may be retained upstream of the blood
permeable element when the capsule is of a size such that it cannot
pass through the blood permeable element. Alternatively, the blood
permeable may be located downstream of the blood permeable element
but retained in place by an attachment means, for example, a hook
or tether, extending from the blood permeable element to the
capsule. In addition, it is contemplated that the capsule may be
wedge-like in shape, such that the narrow end of the wedge may pass
through the element but the larger end contacts the element thereby
to prevent passage of the capsule through the element.
The capsule may comprise either a single hollow fiber or a bundle
of hollow fibers made from a semi-permeable membrane. The
semi-permeable membrane preferably has pores of a size sufficient
to permit the diffusion of the pre-selected molecule therethrough
but yet small enough to exclude the passage of cells therethrough.
The pores preferably are designed to permit the pre-selected
molecule produced by the cells to diffuse directly into the blood
stream passing the hollow fiber while preventing the cells from
migrating out of the hollow fiber and into the systemic
circulation. More specifically, the pores preferably are
dimensioned to allow solutes having a molecular weight of less than
about 150 kilo daltons to pass therethrough while excluding agents
in the blood stream, for example, proteins, specifically,
antibodies and cytolytic factors secreted by lymphocytes, or cells,
specifically, macrophages and lymphocytes, which if allowed to pass
though the pores from the blood stream into the hollow fiber may be
detrimental to the viability of the cells enclosed therein. It is
contemplated that if the preselected molecule has a molecular
weight greater than about 150 kilo daltons then the capsule should
have pores dimensioned to permit the preselected molecule to
diffuse out of the capsule into the blood stream. It should be
noted, however, that the viable cells useful in producing and
secreting preselected molecules having a molecular weight greater
than 150 kilo daltons should be autologous in nature thereby
minimizing a host immune response, humoral and/or cellular,
directed against the cells disposed within the capsule.
Polymers useful in producing biocompatible semi-permeable membranes
of the present invention include, but are not limited to,
polyvinylchloride, polyvinylidene fluoride, polyurethane
isocyanate, polyalginate, cellulose acetate, cellulose diacetate,
cellulose triacetate, cellulose nitrate, polysulfone, polystyrene,
polyurethane, polyvinyl alcohol, polyacrylonitrile, polyamide,
polymethylmethacrylate, polyethylene oxide and
polytetrafluoroethylene. In addition, it is contemplated that
useful semi-permeable membranes may be produced from a combination
of such polymers.
In another preferred embodiment, the viable cells enclosed within
the semi-permeable hollow fiber(s) of the capsule, preferably are
eukaryotic cells, and most preferably are mammalian cells.
Although, the device described herein may comprise cells which
naturally produce and secrete the pre-selected molecule, it is
contemplated that genetically engineered cells, i.e., cells
transfected with, and capable of expressing a nucleic acid encoding
the preselected molecule, may likewise be used in the practice of
the invention.
In another preferred embodiment, the pre-selected molecule
preferably is a protein, and most preferably is a hormone, for
example, erythropoietin or insulin. It is contemplated, however,
that the device may be used to deliver into the systemic
circulation any molecule that can be produced and secreted from a
viable cell. Although single cell types that produce and secrete a
single pre-selected molecule may be used in the invention, it is
understood that cells belonging to a particular cell type that
produce and secrete a plurality of pre-selected molecules likewise
may be used in the practice of the instant invention. Similarly, it
is contemplated that a plurality of cell types, wherein cells
belonging to each cell type produce and secrete different
pre-selected molecules, may be combined in a capsule thereby to
produce a device that delivers a desirable combination of
preselected molecules into the circulation.
In some applications, for example, during hormone replacement
therapy, it is preferable to use cells which produce the
pre-selected molecule in response to an external stimulus. A device
containing such regulated cells therefore produces the pre-selected
molecule when the necessity arises thereby preventing an
overproduction of the pre-selected molecule, which, depending upon
the molecule, may be detrimental to the recipient. However, during
other applications, for example, during replacement therapy of
Factor VIII in Factor VIII deficient hemophilia; Factor IX in
Factor IX deficient hemophilia; or .alpha..sub.1 -anti-trypsin in
.alpha..sub.1 -anti-trypsin deficient emphysema, it is contemplated
that cells which constitutively produce these pre-selected
molecules, may be enclosed in the hollow fibers of the device.
Certain forms of anemias, for example, erythropoietin deficient
anemias caused by end stage renal disease, result from the
inability of the host to produce erythropoietin in an amount
sufficient to induce the production of sufficient numbers of red
blood cells. As a result of this disease, the patient's red blood
cell mass falls thereby lowering the oxygen carrying potential of
the blood. In one preferred embodiment, the invention therefore
provides a device comprising erythropoietin producing cells that
produce erythropoietin in response to the reduced oxygen carrying
capacity of the recipient's blood. The invention permits the
erythropoietin producing cells to be exposed to blood intimately
enough so that full endocrine function of the cells can be realized
effectively. Accordingly, it is contemplated that the implantable
erythropoietin producing and secreting device of the invention may
be useful in the treatment of erythropoietin deficient anemias.
In addition, certain forms of diabetes, for example, diabetes
mellitus, result from an inability of the host to produce insulin
in an amount sufficient to modulate the level of circulating
glucose in the blood stream. In another preferred embodiment, the
invention provides a device comprising insulin producing cells that
produce insulin in response to the level of glucose in the blood.
Accordingly, it is contemplated that the implantable insulin
producing and secreting device of the invention may be useful in
the treatment of insulin dependant forms of diabetes.
In another aspect, the invention provides a method for
percutaneously introducing into a blood vessel of a mammal, a
device for delivering a pre-selected molecule into systemic
circulation. The method comprises the steps of: (a) anchoring a
blood permeable element to an inner wall of an intact blood vessel,
which when anchored permits blood in the vessel to pass
therethrough; (b) introducing at a location upstream of the
anchored element a capsule containing viable cells that produce and
secrete the pre-selected molecule; and (c) permitting the capsule
to contact the element.
In this procedure, the blood permeable element may be introduced
into the blood vessel by means of a catheter. Furthermore, the
capsule may likewise be introduced into the vessel by means of the
same or a different catheter. During such procedures the blood
permeable element and/or the capsule may be introduced by a
catheter into the mammal via a femoral or jugular vein and then
anchored in a natural vein, for example, an inferior vena cava, a
superior vena cava, a portal vein or a renal vein, or
alternatively, anchored in a synthetic vein, for example, a vein
developed from a surgically-developed arteriovenous fistula. It is
contemplated that selection of appropriate sites for introduction
and anchorage of the device is within the expertise of one skilled
in the art.
BRIEF DESCRIPTION OF THE DRAWINGS
The present invention will now be more particularly described with
reference to and as illustrated in, but in no manner limited to,
the accompanying drawings, in which:
FIG. 1 is a side sectional schematic illustration of a first
cell-containing capsule of the type useful in the practice of the
present invention,
FIG. 2 is a side sectional schematic illustration of a second
cell-containing capsule of the type useful in the practice of the
present invention,
FIG. 3 is an illustration depicting a preferred blood permeable
element of the invention,
FIG. 4 is a top plan view of the element of FIG. 3,
FIG. 5 is a cross-sectional schematic view of the device of the
invention disposed within an intact blood vessel, and
FIG. 6A through E are illustrations showing preferred embodiments
of the device of the invention.
In the drawings, like characters in the respective drawings
indicate corresponding parts.
DETAILED DESCRIPTION OF THE INVENTION
In its most general application, the present invention provides an
implantable device for delivering a pre-selected molecule into the
systemic circulation of a mammal. The device of the invention is
adapted for direct implantation into a blood vessel, preferably
using a catheter. After implantation, the device permits the
pre-selected molecule to diffuse out of the device and into the
blood stream of the recipient, which in certain aspects does so in
response to blood parameters, for example, oxygen tension in the
case of erythropoietin-producing cells.
The device comprises two components that interact with one another
when implanted in the recipient. The first component is a blood
permeable element, preferably a cage-like filamentous structure,
that is dimensioned for insertion into the lumen of an intact blood
vessel. Once introduced to a desired location, the element is
anchored in place to an inner wall of the blood vessel typically by
means of hooks or barbs disposed upon the element. The blood
permeable element is designed such that when anchored to the wall
of the blood vessel, the element permits blood in the vessel to
pass therethrough. The second component is a capsule also
dimensioned for insertion into the lumen of the blood vessel. The
capsule comprises a semipermeable housing containing viable cells
which produce and secrete the pre-selected molecule. The capsule is
inserted into the blood vessel upstream of the element. Once
inserted, the capsule may move along the blood vessel with the flow
of the blood until it reaches and contacts the anchored element.
The pre-selected molecule is produced and secreted by the cells
entrapped within the capsule, which after being secreted from the
cells diffuses out of the capsule and into blood passing the
capsule. Upon entry into the blood stream, the pre-selected
molecule is disseminated rapidly throughout the vasculature of the
host. Proper operation of the device requires that it not occlude
the blood vessel, i.e., the implanted device does not prevent
passage of blood through the blood vessel.
The device of the present invention will now be described in
greater detail with reference to the attached drawings, which are
provided for purposes of illustration and are not meant to be
limiting of the scope of the invention. Referring to the drawings,
FIGS. 1 and 2 illustrate schematically capsules 10 useful in the
practice of the invention. In FIG. 1, the capsule 10 comprises a
single hollow fiber made of a semi-permeable membrane 12 which
encloses the viable cells 14. In FIG. 2, the capsule 10 comprises a
semi-permeable membrane 12 enclosing a plurality of semi-permeable
membrane hollow fibers 16 which enclose the viable cells 14. It is
contemplated that semi-permeable membranes 12 and 16 may be defined
by either the same or different polymeric compositions. Methods and
materials for the manufacture of such membranes are known in the
art, and are described below. The viable cells 14 may, or may not,
be attached to an inner surface of a fiber, however, this feature
will depend upon the cell type included in the device. For example,
some cell types grow in an anchorage dependent manner upon a solid
surface while other cell types have no anchorage dependency and
grow in suspension. The choice of cell type, however, is dependent
upon the resulting application.
FIGS. 3 and 4, show a preferred blood permeable element 20 useful
in the practice of the invention. The element 20 comprises a head
26 and a plurality of resilient, typically metallic, legs 22
extending therefrom. The end of the legs distal to the head
comprise hooks or barbs 24 disposed outwardly to engage an inner
wall of the target blood vessel. A variety of such elements based
upon this design are well known in the art, and are described in
more detail below. It is contemplated, however, that other blood
permeable elements based upon other designs, for example, a birds
nest filter described hereinbelow, also may be used in the practice
of the instant invention.
FIG. 5, shows a preferred design configuration for the implantable
device of the invention. The blood permeable element 20 is anchored
to an inner wall 32 of an intact blood vessel 30. The head 26 of
the element 20 having legs 22 extending therefrom biased radially
by spring tension is located in the vessel proximal to the hooks
24. The outwardly disposed legs 22 spread, umbrella-like, to permit
hooks 24 to engage the wall of the blood vessel 30 to prevent
movement of the element in the direction of blood flow (shown by
the arrow). Located upstream of the element 20 is a capsule 10
containing viable cells. The capsule is made of a semi-permeable
membrane that permits oxygen, glucose and other nutrients necessary
for the viability of the encapsulated cells to diffuse from the
blood stream into the lumen of the capsule while permitting cell
metabolites, for example, the pre-selected molecule and cellular
waste products, to diffuse out of lumen of the capsule into the
blood stream.
FIG. 6 shows a variety of configurations believed to be useful in
the practice of the invention. FIG. 6A shows a single capsule 10
comprising a bundle of hollow fibers held together by means of end
caps 40. The single capsule is disposed within a single blood
permeable element 20. Assuming that the hooks or barbs 24 are
embedded into the inner wall of a blood vessel, then capsule 10 is
retained in position within the blood vessel upstream of the blood
permeable element by contact with the blood permeable element 20.
FIG. 6B is essentially the same as FIG. 6A except that two capsules
10 are disposed within and in contact with a single blood permeable
element 20. FIG. 6C shows a single capsule 10 retained within and
in contact with a blood permeable element 20. Part of capsule 10
contacting the blood permeable element 20 is disposed within a
biocompatible gel 50, for example, an autologous blood dot, thereby
to optimize capture of the capsule 10 by the blood permeable
element 20. FIG. 6D shows two capsules 10 retained in place by a
blood permeable element 20. The capsules 10 contact the blood
permeable element 20 by means of hooks or tethers 60. Assuming that
barbs 24 of the blood permeable element 20 are embedded into an
inner wall of a blood vessel, then the capsules 10 would be located
downstream of the blood permeable element. FIG. 6E shows two
capsules 10 held in place by means of two blood permeable elements
20. This type of configuration may be particularly useful when a
large number of cells may be required to produce the preferred
dosage of preselected molecule and, therefore, long capsules 10 are
required to accommodate the large number of viable cells.
The Blood Permeable Element
As mentioned above, the art is replete with blood permeable
elements useful in the practice of the instant invention. Useful
blood permeable elements are characterized by their ability to be
anchored within the lumen of a blood vessel without occluding or
preventing blood flow through the blood vessel.
Blood permeable elements useful in the practice of the invention
are commercially available and are marketed as embolism or blood
clot anti-migration filters. These antimigration filters are used
routinely by medical practitioners to prevent the migration of
potentially life threatening blood clots within the vasculature.
Blood clot anti-migration filters typically are designed to be
implanted and anchored within the lumen of a blood vessel. When
implanted, the anti-migration filters permit blood in the vessel to
pass while simultaneously trapping blood clots.
A variety of blood clot anti-migration filters useful in this
invention are known in the art and are commercially available. For
example, currently preferred blood clot anti-migration filters
described in U.S. Pat. Nos. 4,817,600 and 5,059,205, referred to in
the art as Greenfield.RTM. filters and available from
Medi.Tech.RTM., Boston Scientific Corporation, Watertown, Mass, are
particularly well suited to the practice of the invention. The
cone-shaped Greenfield.RTM. vena cava filters are designed to
provide maximal entrapment area for trapping blood clots while
maintaining patency of the blood vessel after trapping emboli. For
example, the geometry of the cone permits filling to 80% of its
depth before the cross-sectional area is reduced by 64%, and that
at least 80% of the depth of the filter can be filled without
development of a significant pressure gradient across the filter.
The spacing between the six legs of the Greenfield.RTM. vena cava
filters ensures the trapping of emboli greater than 3 mm
(Greenfield et al. (1989) "Venous Interruption" Chapter 68, pp.
929-939 in "Haimovici's Vascular Surgery Principles and Techniques,
Third Edition," Appleton and Lange, Norwalk, Connecticut/San
Mateos, Calif.). Accordingly, the filters may be able to capture
capsules greater than 3 mm in diameter.
Other useful blood clot anti-migration filters useful in the
invention are described in U.S. Pat. Nos. 4,781,177; 4,494,531;
4,793,348; 5,152,777; 5,350,398; and 5,383,887, the disclosures of
which are incorporated herein by reference. Also, it is
contemplated that other blood clot anti-migration filters, such as
those described in Greenfield (1991) in "Vascular Surgery, A
Comprehensive Review " Moore, ed. W. B. Saunders Co., Philadelphia,
London, Toronto, Montreal, Sydney, Tokyo pp. 669-679, including:
Nitinol filters; Gunther filters; Venatech filters; Amplatz
filters; and birds nest filters, likewise may be useful in the
practice of the invention. Because of the inherent properties of
blood clot anti-migration filters, namely their design for
introduction and anchorage within the lumen of a blood vessel, and
further that, when anchored in the vessel, the filters permit blood
in the vessel to pass therethrough, makes them desirable as blood
permeable elements of the invention. It is contemplated, however,
that blood permeable elements other than the ones described herein
but having the aforementioned characteristics also may be useful in
the practice of the instant invention.
Capsule Design
The implanted drug delivery device of the invention may be capable
of delivering a pre-selected drug over a prolonged period of time,
preferably in range of months to years. It is contemplated,
however, that exhausted cell capsules, i.e., wherein a substantial
fraction of cells within the capsule are no longer viable or no
longer secrete the preselected molecule, may be retrieved from the
recipient and replaced with new capsules containing fresh cells
that produce and secrete the preselected molecule.
The capsule may comprise either a single hollow fiber (as shown in
FIG. 1) or a plurality of hollow fibers (as shown in FIG. 2). The
number of fibers depend upon a set of variables that may be
determined without undue experimentation. One variable, for
example, includes the productivity of the cells to be incorporated
into the device. It is appreciated that if a first cell type
produces and secretes more pre-selected molecule than a second cell
type, then fewer cells of the first type will be needed to produce
the same amount of pre-selected molecule. Other variables include:
the amount of the pre-selected molecule necessary to produce the
desired therapeutic effect in the recipient; the nutritional
requirements of the cells; the time over which the cells remain
viable after implantation; and the density to which the cells can
grow without losing viability. Once these variables have been
established, then the practitioner by judicious choice of cell type
and hollow fiber geometry may select the appropriate cell type to
remain viable in the host and produce the desired amount of
preselected molecule for the longest period of time.
Because the device permits delivery of the pre-selected molecule
over prolonged periods of time, an important consideration,
therefore, is the development of capsules which maintain the
viability of the cells enclosed therein after implantation into the
host. It is understood that a variety of factors, for example: the
supply of oxygen and nutrients to the cells in the capsule; the
removal of waste products from the cells in the capsule; the
minimization of host immune responses directed against the cells in
the capsule; the proliferative activity of the cells; and whether
cells located at the center of the capsules are susceptible to
pressure necrosis, all of which may influence the design and
preparation of a cell containing capsule.
Since the transport of oxygen may become a limiting factor for the
viability and function of implanted cells, the geometry of the
hollow fibers must be chosen with care to maintain adequate oxygen
delivery. It is believed that the transport of oxygen from the
lumen of the blood vessel to the cells enclosed within the capsule
occurs almost exclusively by diffusion. Studies have shown that, in
order to maintain the viability of cells excluded from the blood
stream or a blood supply, the cells preferably are located within a
critical diffusion distance of about 500 .mu.m, more specifically
about 300 .mu.m from the blood supply. For example, direct
measurement of the dissolved oxygen levels in mammalian thoracic
aortas with oxygen electrodes have shown that the level of
dissolved oxygen in the arterial wall approaches a nadir of 25 mm
Hg approximately 300 .mu.m from the blood lumen (Buerk et al.
(1982) Am. J. Physiol. 243: H948-H958). In order to ensure optimal
aeration conditions, it is contemplated therefore that the hollow
fibers containing the cells should have an internal diameter
preferably less than about 1000 .mu.m (1.0 mm), and most preferably
less than about 500 .mu.m (0.5 mm). It should be noted that cells
that have a low metabolic activity, and therefore low oxygen
demand, for example, myoblasts may remain viable in hollow fibers
having internal diameters exceeding about 500 .mu.m, however, cell
types with high metabolic activities preferably are entrapped
within hollow fibers having internal diameters of about 200 .mu.m.
Furthermore, it is contemplated that the optimal capsule diameter
for a preselected cell type may be determined without undue
experimentation using the methodolgies described hereinbelow
In addition to adequate aeration, it is important that the
encapsulated cells obtain sufficient amounts of essential nutrients
from the blood supply to remain viable. It is believed that oxygen
diffusion is the most important aspect in maintaining cell
viability and, therefore, once the geometry of a hollow fiber has
been optimized for oxygen transport, then the hollow fiber
inherently will be able to permit the diffusion of adequate amounts
of nutrients into the lumen of the capsule from the blood stream.
Similarly, such a geometry is contemplated also to permit diffusion
of cell metabolites, including, waste products and the pre-selected
molecule, out of the hollow fiber and into the blood stream.
The hollow fibers preferably are produced from a semi-permeable
membrane having pores dimensioned to permit the diffusion of oxygen
and nutrients into the lumen of the hollow fiber while permitting
the efflux of cellular waste products and the pre-selected molecule
out of the hollow fiber. In addition, the pores preferably are
dimensioned to exclude the passage of cells therethrough.
Accordingly, the pores are designed to prevent migration of the
viable cells from the lumen of the hollow fiber into the blood
steam, thereby maintaining the implanted cells at a single location
in the host to facilitate their subsequent removal if or when
necessary. The pores also are designed to prevent the influx of the
hosts immune cells, for example, macrophages and lymphocytes, which
if allowed to enter the lumen of the hollow fibers may be
detrimental to the viability of the cells enclosed therein. The
membrane, therefore, provides an immuno-priviledged environment
that protects cells enclosed therein from an immune response. This
may be an important consideration if the implanted cells are non
autologous in nature.
In addition, it is contemplated that although the pores should be
large enough to permit the exit of the pre-selected molecule, the
pores preferably should exclude molecules, for example, antibodies
and cytotoxic cytokines, having a molecular weight greater than
about 150 kD. It is contemplated that exclusion of host antibodies
and cytokines may enhance the longevity of the viable cells
following implantation of the device into the host. As a result of
the preferred pore exclusion size, it is contemplated that the
hollow fibers are adapted to permit the efflux of pre-selected
molecules having a molecular weight of smaller than 150 kD. Pore
size is an important consideration if the cells entrapped within
the capsule are not autolgous cells. Accordingly, it is appreciated
that if the preselected molecule has a molecular weight exceeding
150 kilo daltons, for example, Factor VIII which has a molecular
weight of about 330 kilo daltons, then the cells that produce and
secrete the preselected entrapped within the hollow fiber should be
autogenic in nature thereby minimizing any immune response directed
against and therefore to extend the longevity of the viable cells.
However, if the preselected molecule has a molecular weight less
than 150 kilo daltons, for example, Factor IX (about 56 kilo
daltons), .alpha..sub.1 anti-trypsin (about 52 kilo daltons) and
erythropoietin (about 36 kilo daltons), then it is anticipated that
any cell type may be entrapped with the hollow fiber, although
autogenic cells are preferred.
The hollow fibers comprising, or for incorporation within, the
capsule may be produced from biocompatible polymers which include,
but are not limited to, polyvinylchloride, polyvinylidene fluoride,
polyurethane isocyanate, polyalginate, cellulose acetate, cellulose
diacetate, cellulose triacetate, cellulose nitrate, polysulfone,
polystyrene, polyurethane, polyvinyl alcohol, polyacrylonitrile,
polyamide, polymethylmethacrylate, polyethylene oxide,
polytetrafluoroethylene or copolymers thereof. A summary of
currently available hollow fibers, including methods of manufacture
and the names of commercial suppliers, is set forth in Radovich
(1995) "Dialysis Membranes: Structure and Predictions" Contrib
Nephrol., Basel, Karger, 113:11-24, the disclosure of which is
incorporated herein by reference. In addition,
polytetrafluorethylene polymer hollow fibers are available
commercially from Impra, Inc., Tempe, Ariz. or W. L. Gore and
Associates, Flagstaff, Ariz.
If enough cells can be implanted in a single hollow fiber to
produce a desirable level of the pre-selected molecule in the blood
stream then the capsule of the invention, preferably will contain a
single hollow fiber (see, for example, FIG. 1 ). Alternatively, if
the requisite number of cells cannot be incorporated into a single
hollow fiber then the appropriate number of cells may be entrapped
within a bundle of hollow fibers wherein bundle of fibers are
further encapsulated within a second macroporous outer membrane
(see, for example, FIG. 2). The porous outer membrane preferably
defines pores that do not affect the diffusion rates of nutrients
and cell metabolites into, and out of the cell-containing hollow
fibers. The purpose of the outer membrane is to hold the bundle of
fibers together and not to limit diffusion of oxygen and nutrients
into the hollow fibers or the diffusion of waste products, i.e.,
carbon dioxide, and the pre-selected molecule out of the hollow
fibers. In such configurations, the resulting bundles of hollow
fibers usually will have an external diameter sufficient to permit
entrapment by the capsule of the blood permeable element. In
addition, a bundle of hollow fibers may be held together by end
caps (see, for example, end caps 40 in FIG. 6A). Alternatively, the
hollow fibers may be encased within a biocompatible gel, for
example, an autologous blood clot prepared from blood extracted
from the intended recipient, thereby to produce a plug that may be
captured by the blood permeable element (see, for example, plug 50,
in FIG. 6C). In addition, it is contemplated, that even if the
capsules are too small to be captured by the blood permeable
element then they may be held in contact with the blood permeable
element means of a hook or tether (see, for example, tether 60 in
FIG. 6D). It is contemplated that the optimal configuration for
each blood permeable element and capsule may be determined without
undue experimentation by the skilled practitioner.
Viable Cells.
It is contemplated that a variety of cell types may be used in the
practice of the instant invention. The cells preferably are
eukaryotic cells and most preferably are mammalian in origin.
Furthermore, the implanted cells most preferably are autogenic,
i.e., the implanted cells are derived from the intended recipient.
However, as discussed above, because the cells of the invention may
be enclosed in an immuno-priviledged environment within a
semipermeable membrane, for example, when the preselected molecule
has a molecular weight less than 150 kilo daltons, it is
contemplated that allogeneic cells, i.e., cells derived from an
another individual within the same species as the intended
recipient, or alternatively xenogeneic cells, i.e., cells derived
from a species other than the species of the intended recipient,
may be used in the practice of the invention.
The cells incorporated within the device preferably are isolated,
established cells or cell lines that produce and secrete the
pre-selected molecule of interest. Such cells or cell lines usually
are isolated by standard cell culture and screening procedures well
known and thoroughly documented in the art. Reviews discussing such
conventional culture and screening procedures include, for example,
"Tissue Culture, Methods and Applications" (1973) Kruse and
Paterson, Eds., Academic Press, New York, San Francisco, London;
"Culture of Animal Cells, A Manual of Basic Technique," Second
Edition (1987) Freshney, Ed., Wiley-Liss, New York, Chichester,
Brisbane, Toronto, Singapore; "Cell Biology, A Laboratory Handbook"
(1994) Celis, Eds., Academic Press; and "Control of Animal Cell
Proliferation" (1985) Boyton and Leffert, Eds., Academic Press.,
the disclosures of which are incorporated herein by reference.
Although the cells or cell lines of interest preferably are
isolated from the recipient and expanded by standard cell culture
methodologies prior to implantation, it is contemplated that useful
cells or cell lines may be isolated from individuals of the same
species other than the intended recipient. Alternatively, useful
cells or cell lines may be isolated from individuals belonging to
other species, i.e., of porcine, murine, equine, bovine, simian,
canine or feline origin. For example, isolated fetal porcine
ventral mesencephalon cells that produce and secrete dopamine have
been implanted into human brain tissue to alleviate the symptoms
associated with Parkinson's disease (Lindwall et al. (1990) Science
575-577).
To be useful, the cells should produce and secrete the pre-selected
molecule of interest either constitutively or in response to
ambient conditions. The practitioner by judicious choice of cells
and cell lines may produce an implantable drug delivery device for
the treatment of a variety of diseases. It is understood, however,
that the cell type will depend upon the disease or symptom to be
treated. For example, in order to produce a device suitable for the
treatment of erythropoietin deficient anemia, the practitioner may
incorporate erythropoietin producing cells into the device of the
invention. Although any cell that produces erythropoietin may be
used, it is believed that optimal cell types produce and secrete
erythropoietin responsive to their environment, i.e., the level of
dissolved oxygen in the blood stream. For example, cell lines that
produce and secrete erythropoietin in response to variations in the
level of dissolved oxygen have been isolated and characterized. See
for example, U.S. Pat. No. 4,393,133 and Goldberg et al. (1987)
Proc. Natl. Acad. Sci. USA. 84: 7972-7976, the disclosures of which
are incorporated herein by reference. Accordingly, the
incorporation of any cell or cell line that produces erythropoietin
in response the oxygen carrying potential of the blood may be
useful in the practice of the invention.
Similarly, a device containing insulin producing cells may be used
in the treatment of insulin dependent diabetes mellitus. Cells
having utility in such a device, preferably are isolated from
either healthy individuals of the same species as the recipient, or
from healthy members from other species, i.e., mammals of porcine,
bovine, equine or simian origin. Methods for isolating, screening,
and culturing insulin producing Islets and dispersed Beta cells as
well as insulin producing cell lines are well known and thoroughly
discussed in the art. See, for example, Lacy et al. (1976) Diabetes
25: 585-594; Wollheim et al. (1990) Methods in Enzymology 192:
188-223; and Wollheim et al. (1990) Methods in Enzymology 192:
223-235, the disclosures of which are incorporated herein by
reference.
In addition to the use of naturally occurring cells or cell lines
that produce and secrete the molecule of interest, it is
contemplated that cells "tailor made" by conventional recombinant
DNA methodologies may be engineered to produce and secrete a
desired pre-selected molecule or a combination of such molecules.
The processes for manipulating, amplifying, and recombining nucleic
acids encoding a pre-selected molecule of interest generally are
well known in the art, are therefore, are not described in detail
herein. Methods for identifying and isolating genes encoding a
pre-selected molecule are also well understood, and are described
in the patent and other literature.
Briefly, the production of DNAs encoding pre-selected molecules of
interest is performed using known techniques involving the use of
various restriction enzymes which make sequence specific cuts in
DNA to produce blunt ends or cohesive ends, DNA ligases, techniques
enabling enzymatic addition of sticky ends to blunt-ended DNA,
construction of synthetic DNAs by assembly of short or medium
length oligonucleotides, cDNA synthesis techniques, polymerase
chain reaction (PCR) techniques for amplifying appropriate nucleic
acid sequences from libraries, and synthetic probes for isolating
genes encoding the molecule of interest. Various promoter sequences
from bacteria, mammals, or insects to name a few, and other
regulatory DNA sequences used in achieving expression, and various
types of host cells are also known and available. Conventional
transfection techniques, and equally conventional techniques for
cloning and subcloning DNA are useful in the practice of this
invention and known to those skilled in the art. Various types of
vectors may be used such as plasmids and viruses including animal
viruses and bacteriophages. The vectors may exploit various marker
genes which impart to a successfully transfected cell a detectable
phenotypic property that can be used to identify which of a family
of clones has successfully incorporated the recombinant DNA of the
vector.
One method for obtaining DNA encoding the molecule of interest may
be isolated from libraries of nucleic acids, for example, by colony
hybridization procedures such as those described in Sambrook et al.
eds. (1989) "Molecular Cloning", Coldspring Harbor Laboratories
Press, N.Y., and/or by PCR amplification methodologies, such as
those disclosed in Innis et al. (1990) "PCR Protocols, A guide to
methods and applications ", Academic Press, the disclosures of
which are incorporated herein by reference. The nucleic acids
encoding the molecule of interest, once isolated, may be integrated
into an expression vector and transfected into an appropriate host
cell for protein expression. Useful prokaryotic host cells include,
but are not limited to, E. coli, and B. Subtilis. Useful eukaryotic
host cells include, but are not limited to, yeast cells, insect
cells, myeloma cells, fibroblast 3T3 cells, monkey kidney or COS
cells, chinese hamster ovary (CHO) cells, mink-lung epithelial
cells, human foreskin fibroblast cells, human glioblastoma cells,
and teratocarcinoma cells.
The vector additionally may include various sequences to promote
correct expression of the recombinant protein, including
transcriptional promoter and termination sequences, enhancer
sequences, preferred ribosome binding site sequences, preferred
mRNA leader sequences, preferred protein processing sequences,
preferred signal sequences for protein secretion, and the like. The
DNA sequence encoding the gene of interest also may be manipulated
to remove potentially inhibiting sequences or to minimize unwanted
secondary structure formation.
Expression of the engineered genes in eukaryotic cells requires
cells and cell lines that are easy to transfect, are capable of
stably maintaining foreign DNA with an unrearranged sequence, and
which have the necessary cellular components for efficient
transcription, translation, post-translation modification, and
secretion of the protein. In addition, a suitable vector carrying
the gene of interest also is necessary. DNA vector design for
transfection into mammalian cells should include appropriate
sequences to promote expression of the gene of interest as
described herein, including appropriate transcription initiation,
termination, and enhancer sequences, as well as sequences that
enhance translation efficiency, such as the Kozak consensus
sequence. Preferred DNA vectors also include a marker gene and
means for amplifying the copy number of the gene of interest. A
detailed review of the state of the art of the production of
foreign proteins in mammalian cells, including useful cells,
protein expression-promoting sequences, marker genes, and gene
amplification methods, is disclosed in Bendig (1988) Genetic
Engineering 7: 91-127.
The various cells, cell lines and DNA sequences that can be used
for mammalian cell expression of the pre-selected molecule are well
characterized in the art and are readily available. Other
promoters, selectable markers, gene amplification methods and cells
also may be used to express the proteins of this invention.
Particular details of the transfection, and expression protocols
are well documented in the art and are understood by those having
ordinary skill in the art. Further details on the various technical
aspects of each of the steps used in recombinant production of
foreign genes in mammalian cell expression systems can be found in
a number of texts and laboratory manuals in the art, such as, for
example, F. M. Ausubel et al., ed., "Current Protocols in Molecular
Biology", John Wiley & Sons, New York, (1989), the disclosure
of which is incorporated by reference herein.
For example, it is contemplated that useful Factor VIII producing
cells may be prepared using conventional recombinant DNA and cell
culture methodologies, and used in the treatment of Hemophilia A.
For example, researchers have exploited the MFG retroviral vector
system to transfer of Factor VIII cDNA into murine and human cells
(primary and established cell lines). The resultant cells exhibit
high levels of Factor VIII production, and that the Factor
VIII--secreting cells upon transfer into immune-deficient mice
produce substantial levels of Factor VIII in the plasma. See, for
example, Dwarki et al. (1995) Proc. Natl. Acad. Sci. USA. 92:
1023-1027, the disclosure of which is incorporated herein by
reference. Likewise, Factor IX producing cells may be prepared and
used in the treatment of Hemophilia B. Researchers have used
retroviral vectors to introduce human or canine Factor IX cDNAs
into cultured primary murine myoblasts, canine myoblasts and into
an established murine myoblast cell line. In all cases, the
resulting stably transfected cells produce biologically active
Factor IX in culture and secreted detectable amounts into culture
medium before and after differentiation into myotubes. See, for
example, Roman et al. (1992) Somatic Cell and Molecular Genetics
18: 247-248, the disclosure of which is incorporated herein by
reference.
In addition, .alpha..sub.1 -anti-trypsin producing cells may be
prepared and used in the treatment of emphysema. For example, a
retroviral vector has been used to insert human .alpha..sub.1
-anti-trypsin cDNA into the genome of mouse fibroblasts thereby to
create a clonal population of mouse fibroblasts that produce and
secrete human .alpha..sub.1 -anti-trypsin. See, for example, Garver
et al. (1987) Science 237: 762-764, the disclosure of which is
incorporated herein by reference. Similarly, hepatocytes removed
from the livers of experimental animals have been modified
following transfection with a retroviral vector containing
.alpha..sub.1 -anti-trypsin DNA. The resulting hepatocytes, upon
infusion into the portal circulation of the recipient produce and
secrete detectable levels of .alpha..sub.1 -antitrypsin for up to a
month. See, for example, Crystel (1992) Am. J. Med. 92 (Suppl 6A)
6A 445-6A 525; and Kan et al.; (1992) Proc. Natl. Acad. Sci. USA
89: 89-93, the disclosures of which are incorporated herein by
reference.
In addition, recombinant erythropoietin secreting cell lines may be
produced and used in the treatment of erythropoietin deficient
anemia. For example, myoblast clones carrying a 1.34 kilo base
human erythropoietin cDNA stably produce and secrete high levels of
functional human erythropoietin, and upon transfer into murine
models have been shown to be effective in increasing the hematocrit
levels for at least twelve weeks. See, for example, Hamamori et al.
(1995) J. Clin. Invest. 95: 1808-1813, the disclosure of which is
incorporated herein by reference.
Furthermore, it is contemplated that cells capable of expressing
and secreting aldolase B may be used in the treatment of hereditary
fructose intolerance; glucose-6-phosphatase producing cells may be
useful in the treatment of glycogen storage disease Type I; acid
.alpha.-glucosidase producing cells may be useful in the treatment
of glycogen storage disease Type II; amylo-1,6-glycosidase
producing cells maybe useful in the treatment of glycogen storage
disease Type III; muscle phosphorylase may be useful in the
treatment of glycogen storage disease Type IV;
galactose-1-phosphate uridyl transferase producing cells may be
useful in the treatment of galactosemia; phenylalanine hydroxylase
producing cells may be useful in the treatment of phenylketonuria;
tyrosine aminotransferase producing cells may be useful in the
treatment of tyrosenemia; adenosine deaminase producing cells may
be useful in the treatment of combined immunodeficiency disease;
phorphobilinogen deaminase and URO decarboxylase producing cells
may be useful in the treatment of porphyria; .alpha.-iduronidase
and induronate sulfatase producing cells may be useful in the
treatment of mucopolysaccharidoses; sphingomyelinase producing
cells may be useful in the treatment of Neimann-Pick disease;
glucocerebrosidase producing cells may be useful in the treatment
of Gauchers syndrome; .alpha.-galactosidase producing cells may be
useful in the treatment of Fabry's disease; von Willebrand Factor
producing cells may be useful in the treatment of von Willebrand's
disease; and antithrombin producing cells may be useful in the
treatment of antithrombin deficiency. All of the aforementioned
cells and cell types may be produced using conventional recombinant
DNA and equally conventional cell culture methodologies. It is
understood that the aforementioned examples are not meant to be
limiting in any way, because, it is contemplated that any cell or
cell line that produces and secretes a pre-selected molecule useful
in alleviating the symptoms associated with a particular condition,
once isolated, may be used in the practice of the invention.
The penultimate expression vehicles for expression of the
pre-selected molecule preferably are cells of eukaryotic, most
preferably mammalian origin. Eukaryotic cells may be better suited
to the development of regulated cells that produce and secrete the
pre-selected molecule in response to an external stimulus. It is
contemplated, however, that under particular circumstances, for
example, where no regulation mechanism is required and the
pre-selected molecule can be produced constitutively, engineered
prokaryotic cells may also be useful in the practice of the
invention.
For example, under particular circumstances, for example, during
the use of polysulfone hollow fibers, the formation or capture of
thrombii on or around the device may affect the flow of blood
around the device and/or the diffusion of nutrients or metabolites
into or out of the hollow fibers. Under, these circumstances, it is
contemplated that a cell type that constitutively produces and
secretes an anti-coagulant, for example, tissue plasminogen
activator, streptokinase, urokinase, hirudin or the like, into the
blood stream also may be included within a hollow fiber. Therefore,
the artisan may produce a device containing cells that either on
their own or in combination produce an anticoagulant in addition to
another therapeutic protein.
By way of example, it is contemplated that a gene encoding the
anti-coagulant protein huridin may be introduced into a host cell
by conventional gene transfer methodologies. The local production
of hirudin by endothelial cells may prove especially attractive in
preventing thrombosis at vascular sites. Studies have shown that
hirudin is an effective anticoagulant in vivo and is superior to
heparin in experimental animal models of thrombosis following
arterial injury (Haskel et al. (1991) Circulation 83: 1048-1056;
Heras et al. (1990) Circulation 82: 1476-1484). For example, the
hirudin encoding gene may be isolated by standard PCR protocols and
ligated into a retroviral expression vector, for example pMFG
Moloney murine leukemia tumor virus (Dranoff et al. (1993) Proc.
Natl. Acad. Sci. USA 90: 3539-3542) downstream of a nucleic acid
sequence encoding a signal sequence for vonWillebrand factor (vWF).
The vector subsequently may be packaged into .phi.-crip, an
amphotropic, replication defective recombinant retrovirus (Danos et
al. (1988) Proc Natl. Acad. Sci. USA 85: 6460-6464). Endothelial
cells, i.e., rabbit endothelial cells or human umbilical vein
endothelial cells, subsequently may be infected with the
recombinant retrovirus, which results in the transfer of the
hirudin gene into the genome of the endothelial cell. The
transfected endothelial cells subsequently constitutively produce
and secrete the recombinant hirudin gene product.
Preparation of the Capsule
Once appropriate cells or cell lines that produce and secrete the
pre-selected molecule have been isolated or produced, the cells or
cell lines thereafter are cultured in the hollow fibers which are
used to produce the capsule. The preferred method for introducing
the cells into, as well as culturing the cells within the hollow
fibers is by means of a commercial bioreactor. A list of
manufacturers of commercially available bioreactors is set forth in
"Genetic Engineering News", Feb. 1, 1995, the disclosure of which
is incorporated by reference. Preferred bioreactors useful in the
practice of the invention include: Tricentric.TM. and MabMax.TM.
bioreactors from Setec, Livermore, Calif.; Cell-Pharm.TM. Micro
Mouse.TM. from Unisyn Technologies, Milford, Mass; Cellmax.TM. Quad
from Cellco, Inc., Germantown, MD; and Vitafiber II Hollow Fiber
Cell Culture System from Amicon, Inc., W. R. Grace and Co.,
Beverly, Mass.
Typically, a suspension of cells that produce and secrete the
preselected molecule in cell culture medium is seeded into a
bioreactor by infusing cell containing medium into the hollow
fibers of the bioreactor. This step results in the capture of the
cells within the hollow fibers. Thereafter, the cells are cultured
under the optimal culture conditions for the particular cell type
in accordance with the manufacturers instructions.
The resulting hollow fibers subsequently may be implanted either
alone or as a bundle of hollow fibers in combination with the blood
permeable element into the vasculature of the recipient. Methods
for implantation are discussed below.
Implantation of the Device
The device of the invention preferably is inserted into the
vasculature of the host by a non-surgical or minimally invasive
surgical procedure. More specifically, it is contemplated that the
devices of the invention may be introduced by a variety of
catheter-based devices that have been developed for implanting
blood clot anti-migration filters into the vasculature.
For example, U.S. Pat. Nos. 5,147,379 and 3,952,747, and
International Patent Application Ser. No. PCT/US92/08366, the
disclosures of which are incorporated herein by reference, describe
catheter-based devices and methods for implanting blood clot
anti-migration filters into the vasculature of a recipient.
Typically, the catheter-based filter insertion instruments
comprise: a carrier for supporting a blood clot anti-migration
filter in a collapsed, compact state; an ejector mechanism, usually
located within the carrier for ejecting the filter at the
pre-selected site; and an elongated, flexible tube connected to the
carrier for advancing the carrier along the blood vessel to the
pre-selected location. Once introduced to the preferred location in
the blood vessel, the filter is ejected from the carrier. When self
opening and implanting filters are used, the filter is simply
ejected from the carrier, whereupon the filter anchors itself to
the wall of the blood vessel. If, however, a filter to be manually
opened and anchored is used, then the insertion instrument may
contain additional means for effecting such opening and anchorage
steps. It is contemplated, however, that the skilled practitioner
may insert the commercially available blood clot anti-migration
filter using filter insertion instruments and methods as
recommended by the manufacturer of the filter.
For example, in a preferred embodiment, the device of the invention
comprises a filter element, such as the ones described in U.S. Pat.
Nos. 4,817,600 and 5,059,205, that are known as Greenfield.RTM.
vena cava filters and available commercially from Medi-tech.RTM.,
Boston Scientific Corp., Watertown, Mass. The Greenfield filters
are purchased pre-loaded into a introducer catheter. Accordingly,
it is contemplated that a physician may implant a filter element,
such as the ones described in U.S. Pat. Nos. 4,817,600 and
5,059,205 in accordance with the Meditech.RTM.'s "Instructions For
Use" that are provided with the filters. Accordingly,
Medi-tech.RTM.'s "Instructions for Use" are incorporated herein by
reference, and described below.
Briefly, the filters, typically are inserted through the internal
jugular or femoral vein by percutaneous puncture. During
percutaneous insertion, and after a conventional cavogram, either
the jugular or the femoral vein is punctured with a needle and a
guide wire inserted into the vessel through the needle. Then, a
combined sheath/dilator unit is pushed into the vein over the guide
wire until the end of the sheath is located beyond the implant
site. While holding the sheath in place, the dilator and guidewire
are removed, leaving the sheath behind. The sheath acts as an
access to permit the insertion of the introducer catheter, which
contains a carrier holding the filter. The sheath is flushed with
sterile heparinized saline to prevent potential thrombus formation
within the sheath which may occur during insertion of the
introducer catheter. The introducer catheter is advanced into, but
not beyond the end of, the sheath until the tip of the filter
carrier capsule is positioned adjacent to the implant site. Then,
the sheath is retracted onto the introducer catheter until the
carrier capsule is completely exposed. Then, the filter is pushed
out of the carrier capsule by a pusher mechanism, whereupon the
legs of the filter spring outward and engage the inner wall of the
blood vessel thereby anchoring the filter in position. Once the
filter has been ejected and anchored in the blood vessel, a capsule
or capsules containing the viable cells likewise may be introduced
via the same catheter into the blood vessel at a position upstream
of the anchored filter. Then, the introducer catheter is removed
from the vessel through the sheath. Once the introducer catheter
has been removed, the sheath is also removed, and the puncture site
compressed until homeostasis is achieved.
It is understood that the preferred location for implantation of
the device within the systemic circulation, however, may depend
upon the intended use of the device. For example, in some
situations it is contemplated that it may be desirable to introduce
the devices via the femoral or jugular veins and then anchor the
blood permeable element at a location within a natural vein, such
as, an inferior vena cava, a superior vena cava, a portal vein or a
renal vein. Alternatively, the device of the invention may be
anchored in a synthetic vein, such as a vein developed from a
surgically-developed arteriovenous fistula. If, however, the device
is to be used in hormone replacement therapy the physician may
choose to implant the devices at a location downstream of the
natural site of production of the preselected molecule. For
example, .alpha..sub.1 -anti-trypsin typically is produced by liver
hepatocytes; accordingly, it may be desirable to introduce and
anchor an .alpha..sub.1 -antitrypsin producing device of the
invention downstream of the liver, for example, in the hepatic
vein. In addition, as mentioned above, insulin is produced by Beta
cells of the pancreas; accordingly it may be desirable to anchor an
insulin producing device of the invention within the portal vein
downstream of the pancreas.
It is understood, however, that the physicians judgment based upon
clinical circumstances should determine on a case by case basis the
optimal mode for introducing the device as well as the optimal
location for anchoring the device. Such judgments are contemplated
to be within the scope of expertise of the skilled physician.
Practice of the invention will be still more fully understood from
the following examples, which are presented herein for illustration
only and should not be construed as limiting the invention in any
way.
EXAMPLE 1
Growth of Erythropoietin Producing Cells in Culture
Erythropoietin is a hormone that is produced in specialized cells
in the kidney and released into the circulation when the oxygen
delivery to these specialized cells declines. Under hypoxic
conditions resulting from a decline in red blood cell mass, a
decline in oxygen delivery to tissues occurs. Subsequently, the
decline in dissolved oxygen content induces the specialized kidney
cells to increase erythropoietin production thereby stimulating
increased production of red blood cells. Upon return to normal
oxygen delivery with normoxia or increased red cell mass, the
enhanced production of erythropoietin is suppressed, closing the
classic feedback loop. Erythropoietin although being essential for
homeostasis of blood cell mass is deficient in many chronic
diseases, including, for example, chronic renal failure, rheumatoid
arthritis, autoimmune diseases, chronic infections, human acquired
immunodeficiency syndrome and cancer.
Erythropoietin is a 36 kD glycoprotein that comprises a polypeptide
chain 165 amino acids in length (Miyake et al. (1977) J. Biol.
Chera. 252: 5558-5564). The gene for erythropoietin has been
cloned, transfected into Chinese hamster ovary cells, and expressed
to produce active erythropoietin (Lin et al. (1985) Proc. Natl.
Acad. Sci. USA 82: 7580-7585). Researchers have isolated a human
hepatoma derived cell line, called Hep G2, which subsequently was
found to produce and secrete erythropoietin in response to varying
oxygen tensions in the culture media (Goldberg et al. (1987) Proc.
Natl. Acad. Sci. USA 84: 7972-7976). The Hep G2 cell line is
described in U.S. Pat. 4,393,133, incorporated above, and is
available through the American Type Culture Collection (ATCC),
Rockville, Md. under the accession number ATCC HB 8065.
In order to demonstrate oxygen deficiency erythropoietin production
Hep G2 cells were seeded into cell culture dishes and grown in a
RPMI 1640 medium supplemented with 10% fetal calf serum in a
humidified atmosphere of 5% CO.sub.2 at a temperature of 37.degree.
C. After 3-5 days in culture, the level of dissolved oxygen in the
medium was adjusted by altering the oxygen content of the oxygen
supply. Under basal, well oxygenated conditions, the composition of
the air supply contained approximately 21% oxygen, 5% carbon
dioxide, and 74% nitrogen. Under hypoxic conditions, the
composition of the air supply contained approximately 1-2% oxygen,
5% carbon dioxide, and 93-94% nitrogen.
After incubation for three days, the production of erythropoietin
was measured under both well oxygenated and hypoxic conditions
using a Quantikine.TM.IVD.TM. erythropoietin immunoassay kit from R
& D Systems (Minneapolis, Minn). The assays were performed in
accordance with the manufacturers instructions.
Under basal well oxygenated conditions, the Hep G2 cells produced
about 20 mU erythropoietin/10.sup.6 cells over a time period of 24
hours. Under hypoxic conditions, the level of erythropoietin
production increased to about 100-200 mU/10.sup.6 cells over the
same time period.
EXAMPLE 2
Growth of Erythropoietin Producing Cells in Hollow Fibers
Patients suffering from erythropoietin deficient anemia, for
example, in patients suffering from end stage renal disease,
require approximately 10,000 units of recombinant human
erythropoietin per week ("Proceedings from ESRD Patient Management:
Strategieis for Meeting the Clinical and Economic Challenges",
Nissenson, ed.(1993) in Am. J. Kid. Diseases 22(1) Suppl).
Accordingly, the average patient may be estimated to require
approximately 1,000 units of erythropoietin per day. Furthermore,
assuming that under stimulated conditions, Hep G2 produce greater
than 200 mU/10.sup.6 cells over a 24-hour period, it may be
estimated that approximately 10.sup.9 cells may produce sufficient
erythropoietin to maintain appropriate hematocrit level in such
patients.
As the cells of the invention are provided in hollow fibers, it is
an object of this experiment to demonstrate that erythropoietin
producing cells may be grown within a hollow fiber without
compromising cell viability and/or erythropoietin production.
Erythropoietin producing Hep G2 cells (approximately
4.5.times.10.sup.7 /ml) were seeded into hollow fibers,
specifically polysulfone hollow fibers (W. R. Grace and Associates)
and cellulose acetete hollow fibers (Cellco, Inc.), having a
molecular weight cut-off of 50 kilo daltons and internal diameters
of 210, 350 or 510 .mu.m. The hollow fibers were previously lined
with laminin or collagen Type IV. The membranes used in these
experiments had pore sizes that permitted solutes having molecular
weights lower than 50 kD to pass out of the hollow fiber and into
the culture medium. The pore size, therefore, permitted
erythropoietin (36 kilo daltons) to diffuse out of the hollow fiber
and into the surrounding medium while, at the same time, preventing
migration of the Hep G2 cells out of the hollow fiber. After
seeding, the hollow fibers were placed in RPMI 1640 culture medium
supplemented with 10% fetal calf serum and the cells grown under
well oxygenated conditions (21% oxygen) at 37.degree. C. for 2-3
weeks.
After 2-3 weeks in culture, the amount of erythropoietin secreted
into the medium was assayed as described in Example 1. In addition,
the hollow fibers were removed from the culture medium and fixed
for histological examination to assess the presence of cell
necrosis in the hollow fiber as well as the distribution of
cellular messenger RNA (mRNA) encoding the recombinant
erythropoietin within each micro-environment of the hollow fiber.
The histology of the cells was analyzed using standard histological
techniques and the cellular erythropoietin mRNA levels were
assessed using a standard in situ hybridization protocol ("In Situ
Hybridization Histochemistnf" (1990) Chesselet, ed., CRC Press,
Boca Raton, Ann Arbor, Boston).
The Hep G2 cells grown within the hollow fibers having internal
diameters of 210 .mu.m remained viable because the critical
distance for oxygen diffusion was small enough to permit the cells
to derive sufficient oxygen and nutrients from the medium. It was
found, however, that Hep G2 cells grown in fibers having diameters
of 350 .mu.m exhibited necrosis at the center of the hollow fibers.
Accordingly, it appears that the cells in the middle of the hollow
tube became too dense to obtain sufficient nutrients and oxygen
from the surrounding culture medium. The Hep G2 cells grown in
fibers of internal diameters of 510 .mu.m did not suffer from
necrosis but the cell layer attached to the inner wall of the
hollow fiber was about 150 .mu.m thick. It appears that this
distance correlates with the distance over which sufficient
nutrients and oxygen may diffuse to maintain viability of the Hep
G2 cells. Accordingly, it appears that hollow fibers having
internal diameters of 510 .mu.m may be useful in the development of
devices containing Hep G2 cells because the cells may proliferate
until oxygen and nutrient levels become limiting.
EXAMPLE 3
Oxygen Regulation of Erythropoietin Production of Hep G2 Cells In
Hollow Fibers
The aim of this experiment is to demonstrate that erythropoietin
gene expression and protein production can be regulated by the
oxygen tension of the medium surrounding the hollow fibers. This
experiment may be performed using either single hollow fibers or
bundles of hollow fibers to measure how the rate of erythropoietin
production varies with respect to the concentration of oxygen
dissolved in the culture medium.
Single hollow fibers and bundled hollow fibers of varying
diameters, for example, 210 .mu.m or 510 .mu.m collagen treated
polysulfone hollow fibers (Fresenius, USA, Wallnut Creek, Calif.)
having a molecular weight cut-off about 50 kilo daltons) will be
seeded with Hep G2 cells and allowed to grow under optimal
conditions as determined in Example 2. After maximal cell growth
has been attained, the oxygen concentration of the surrounding
growth medium is adjusted by altering the oxygen content of the air
supply. The compositions of the air supply are modified to oxygen
concentrations of 21%; 5%; 3%; 2%; or 1% oxygen. After 24 hours,
the amount of human erythropoietin secreted into the medium is
measured using the procedure described in Example 1.
The cells in each hollow fiber are examined for cell viability to
determine whether cell viability can be maintained under low oxygen
conditions within the surrounding medium. Furthermore, the core of
cells within the hollow fiber are fixed and the erythropoietin mRNA
levels throughout the core determined by the standard in situ
hybridization procedure used in Example 2.
It is anticipated that using this type of experiment, the viability
of the cells and the production of erythropoietin may be measured
to determine how many cells may be introduced into a hollow fiber
and how many hollow fibers must be used in a device of the
invention to produce the desired amount of erythropoietin. Both
single and bundled hollow fiber constructs may be tested to
determine whether scale up affects the oxygen dependent
erythropoietin production of the cells.
EXAMPLE 4
Implantation of Erythropoietin Producing Cells In Vivo
The aim of this experiment is to determine the viability of Hep G2
cells following implantation of hollow fibers containing Hep G2
into the systemic circulation of an animal.
Experimental dogs are anesthetized with ketamine. While
anesthetized, and by means of fluoroscopic guidance, a titanium
Greenfield.RTM. vena cava filter, such as the one described in U.S.
Pat. No. 5,059,205, is introduced by means of a catheter into a
femoral vein. When the filter is positioned correctly within the
inferior vena cava, as determined by fluoroscopy, the filter is
ejected from the insertion device. Once discharged, the filter's
leg hooks instantly secure the filter to the vena cava wall.
Then, Hep G2 containing hollow fibers prepared in accordance with
the optimal conditions found in Example 3 are prepared for
intravenous implantation upstream of the anchored Greenfield vena
cava filter. The hollow fibers are prepared for injection by
encasing them within a fibrin blood clot produced from blood
removed from the host animal. In addition, conventional radioopaque
platinum tags are incorporated into the hollow fibers to ensure
appropriate placement of the fibers and to assist in retrieval of
the fibers after the sacrifice of the animal. Then, the fibrin clot
containing the hollow fibers is administered through the
intravenous catheter upstream of the anchored Greenfield.RTM. vena
cava filter. The fibrin clot containing the hollow fibers is
captured and retained in place within the inferior vena cava by the
filter.
Then, the filter insertion device is removed from the animal and
the animal allowed to recover from anesthesia. The animals
subsequently are observed at varying time intervals after
implantation. After a postoperative period ranging from about 2
weeks to about 6 months the animals are sacrificed and the hollow
fibers retrieved for cell viability analysis. In addition the
levels of erythropoietin mRNA in various micro-environments within
the hollow fiber are determined by in situ hybridization using the
method described in Example 1.
It is anticipated that the implanted cells within the capsule
remain viable and that there are detectable levels of cellular
erythropoietin mRNA production within the implanted cells.
EXAMPLE 5
Implantation of Erythropoietin Producing Cells into Animals
Suffering From Erythropoietin Deficient Anemia
The aim of this experiment is to determine whether a device of the
invention comprising viable Hep G2 cells, following implantation
into a mammal, may ameliorate the symptoms associated with
erythropoietin deficient anemia.
Experimental dogs (1 and 2/3 nephrectomized dogs) are anesthetized
with ketamine. While anesthetized, and by means of fluoroscopic
guidance, a titanium Greenfield.RTM. vena cava filter, is inserted
by means of a catheter via a femoral vein into the inferior vena
cava. When the filter is positioned correctly within the inferior
vena cava, as determined by fluoroscopy, the filter is ejected from
the insertion device and anchored to the wall of the inferior vena
cava.
Then, the Hep G2 containing hollow fibers prepared in accordance
with the optimal conditions found in Example 4 are prepared for
intravenous implantation upstream of the anchored Greenfield.RTM.
vena cava filter. The hollow fibers are prepared for injection by
encasing them within a fibrin blood clot produced from blood
removed from the host animal. In addition, conventional radioopaque
platinum tags are incorporated into the hollow fibers to ensure
appropriate placement of the fibers and to assist in retrieval of
the fibers after the sacrifice of the animal. Then, the fibrin clot
containing the hollow fibers is administered through the
intravenous catheter upstream of the anchored Greenfield.RTM. vena
cava filter. The fibrin clot containing the hollow fibers is
captured and retained in place within the inferior vena cava by the
filter.
The filter insertion device is removed from the animal and the
animal allowed to recover from anesthesia. Thereafter, the hemocrit
and erythropoietin levels are measured at varying time intervals
after implantation. After a postoperative period ranging from about
2 weeks to about 6 months the animals are sacrificed and the hollow
fibers retrieved for cell viability analysis. In addition the
levels of erythropoietin mRNA in various micro-environments within
the hollow fiber are determined by in situ hybridization using the
method described in Example 1. Control experiments wherein a non
cell containing device is implanted into a nephrectomized dog and
wherein a cell containing device is implanted into a normal dog
also will be performed.
It is anticipated that nephrectomized dogs implanted with an
erythropoietin producing device of the invention will exhibit
elevated erythropoietin and hematocrit levels and maintenance of
homeostatis relative to nephrectomized dogs treated with a non cell
containing device. In addition, it is contemplated that cellular
erythropoietin mRNA levels will be higher in cells implanted into
nephrectomized dogs than non-nephrectomized dogs.
EXAMPLE 6
Isolation and Culture of Insulin Producing Cells in Culture.
Insulin is a 5.8 kilo dalton peptide hormone comprising two
polypeptide chains connected by disulfide bonds. Insulin is
produced by specialized cells (Beta cells of the Islets of
Langerhans) located in the pancreas in response to circulating
levels of glucose in the blood stream. In response to elevated
levels of glucose, insulin is produced thereby stimulating glycogen
synthesis in the liver and muscles. The inability of a mammal to
produce insulin in an amount sufficient to maintain euglycemia
results in the condition of diabetes mellitus, which to date has
been controlled by parenteral administration of exogenously
produced insulin.
Methods for isolating and culturing Islets and Beta cells are
thoroughly documented in the art. See, for example, Lacy et at
(1976) Diabetes 25:484-493; Wollheim et al. (1990) Methods in
Enzymology 192: 188-223; and Wollheim et al. (1990) Methods in
Enzymology 192: 223-235.
For example, in a collagenase based procedure, collagen pretreated
tissue pancreatic tissue is excised from the donor mammal. After
collagenase treatment, Islets of Langerhans are filtered through a
filter having approximately 400 .mu.m mesh holes, and patches that
pass through the filter are harvested into a tube containing
Hank's-HEPES-bovine serum albumin (BSA) buffer. Then, the patches
are separated from other cells by density gradient centrifugation
upon a Histopaque 1077 containing gradient. The Islets are
harvested and transferred into a cell culture dish containing
Hank's-HEPES-BSA solution. Upon resuspension, and with the aid of a
dissecting microscope, the Islets are picked up by a siliconized
pasteur pipette, and transferred to a centrifuge tube containing
Hank's solution. The Islets are harvested by centrifugation,
resuspended in RPMI 1640 tissue culture medium supplemented with
antibiotics and 10% fetal calf serum and incubated at 37.degree. C.
in an atmosphere containing 5% carbon dioxide.
After incubation for five days, the level of insulin secretion into
the medium is measured using a radioimmunoassay procedure "Sigma
Immuno-file" provided by Sigma Chemical Co., St. Louis, Mo. for use
with Sigma product number I8510. It is anticipated that the
isolated Islets produce and secrete detectable levels of insulin
into the cell culture medium.
EXAMPLE 7
Growth of Insulin Producing Cells in Hollow Fibers
It has been determined using Islet cells implanted into
pancreatized dogs that about 2.times.10.sup.5 Islet cells maintain
endocrine function in a 20 kg dog. Assuming 10.sup.4 Islet cells
per kilo, it is estimated that a 50 kg human would likely require
about 10.sup.5 Islet cells to maintain endocrine function. Assuming
that hollow fibers having an internal diameter of about 250 .mu.m
are used in the device of the invention, and further that Islet
patches 200 .mu.m in diameter area isolated, it is estimated that
about 250 Islets may be inserted into a 5 cm hollow fiber.
Accordingly, 1000 of such fibers would yield a device containing
about 2.5.times.10.sup.5 Islets.
As the cells of the invention are provided in hollow fibers, an
object of this experiment is to determine whether insulin producing
Islets cells can be grown within a hollow fiber without
compromising cell viability and/or insulin production.
Approximately 200 Islets isolated in Example 6 are seeded into
hollow fibers, specifically, polysulfone hollow fibers (W. R. Grace
and Associates) and cellulose acetete hollow fibers (Cellco, Inc.),
having molecular weight cut-off of 50 kilo daltons and internal
diameters of 210, 350 or 510 .mu.m. The membranes used in these
experiments have pore sizes that permit solutes having molecular
weights lower than 50 kD, and therefore insulin to pass
therethrough while at the same time preventing migration of the
Islets out of the hollow fiber. It should be noted that in order to
produce Islets small enough to be introduced into the 210 .mu.m
hollow fibers, the Islets produced by collagenase treatment should
be filtered through a mesh having a pore size of about 190 .mu.m.
After seeding, the hollow fibers are placed in RPMI 1640 culture
medium supplemented with 10% fetal calf serum and grown at
37.degree. C. for 2-3 weeks.
After 2-3 weeks in culture, the amount of insulin secreted into the
medium is assayed as described in Example 6. In addition, the
hollow fibers are removed from the culture medium and fixed for
histological examination to assess cell viability within the hollow
fiber as well as the distribution of cellular insulin protein
levels within each micro-environment of the hollow fiber. The
histology of the Islets are analyzed using standard histological
methodologies and the cellular insulin protein levels assessed
using a standard immunohistochemical procedures (See, for example,
"Immunocytochemistry, Practical Applications in Pathology and
Biology", Polak and Van Noorden, eds. (1983) Wright PSG, Bristol,
London, Boston, and "Immnunochemical Methods in Cell and Molecular
Biology", Mayer and Walker, eds. (1983) Academic Press, London, San
Diego, New York, Boston, Sydney, Tokyo, Toronto).
It is contemplated that the Islet cells encapsulated within the
hollow fibers remain viable and produce detectable levels of
insulin.
EXAMPLE 8
Glucose Regulation of Insulin Production by Islets in Hollow
Fibers
The aim of this experiment is to demonstrate that insulin
production can be regulated by the level of glucose in the medium
surrounding the hollow fibers. This experiment may be performed
using either single hollow fibers or bundles of hollow fibers to
measure how the rate of insulin production varies with respect to
the glucose level of the culture medium.
Single hollow fibers and bundled hollow fibers of varying
diameters, for example, 210 .mu.m or 510 .mu.m (collagen treated
polysulfone hollow fibers (W. R. Grace and Associates) and
cellulose acetete hollow fibers (Cellco, Inc.), having molecular
weight cut-off of about 20-50 kilo daltons) will be seeded with
Islets and allowed to grow under optimal conditions as determined
in Example 7. After seven days in culture, the culture medium is
replaced with fresh culture medium supplemented with glucose
ranging in concentration from about 100 to about 500 mg/dL. After
24 hours, the amount of insulin secreted into the medium is
measured, using the procedure described in Example 6.
The Islets in each hollow fiber are examined for cell viability to
determine whether cell viability can be maintained under conditions
that simulate in vivo conditions. Furthermore, the core of cells
within the hollow fiber are fixed and insulin protein levels
throughout the core are determined by a standard
immunohistochemical procedure, such as of the type described in
Example 7.
It is anticipated that using this type of experiment, the viability
of the cells and the production of insulin may be measured to
determine how many Islets may be introduced into a hollow fiber and
how many hollow fibers must be used in a device of the invention to
produce the desired amount of insulin. Both single and bundled
hollow fiber constructs may be tested to determine whether scale up
affects glucose dependent insulin production in the Islets.
EXAMPLE 9
Implantation of Insulin Producing Islet Cells In Vivo
The aim of this experiment is to determine the viability of Islet
cells following implantation of hollow fibers containing such cells
into the systemic circulation of an animal.
Experimental dogs are anesthetized with ketamine. While
anesthetized, and by means of fluoroscopic guidance, a titanium
Greenfield.RTM. vena cava filter, is introduced by means of a
catheter into a femoral vein. When the filter is positioned
correctly within the inferior vena cava, as determined by
fluoroscopy, the filter is ejected from the insertion device. Once
discharged, the filter's leg hooks instantly secure the filter to
the vena cava wall.
Then, Islet cell containing hollow fibers prepared in accordance
with the optimal conditions found in Example 8 are prepared for
intravenous administration and implantation within the anchored
Greenfield.RTM. vena cava filter. The hollow fibers are prepared
for injection by encasing them within a fibrin blood clot produced
from blood removed from the host animal. In addition, conventional
radioopaque platinum tags are incorporated into the hollow fibers
to ensure appropriate placement of the fibers and to assist in
retrieval of the fibers after the sacrifice of the animal. Then,
the fibrin clot containing the hollow fibers is administered
through the intravenous catheter upstream of the anchored
Greenfield.RTM. vena cava filter. The fibrin clot containing the
hollow fibers is captured and retained in place within the inferior
vena cava by the filter.
The filter insertion device is removed from the animal, and the
animal is allowed to recover from anesthesia. Subsequently, the
animals subsequently are observed at varying time intervals after
implantation. After a postoperative period ranging from about 2
weeks to about 6 months, the animals are sacrificed and the hollow
fibers retrieved for cell viability analysis. In addition, the
levels of insulin protein production in various micro-environments
within the hollow fiber are determined by an immunohistochemical
procedure.
It is anticipated that the implanted Islet cells encapsuled within
the capsule remain viable and that there are detectable levels of
cellular insulin production within the implanted cells.
EXAMPLE 10
Implantation of Insulin Producing Cells in Animals Suffering From
Insulin Deficient Diabetes
The aim of this experiment is to determine whether a device of the
invention comprising viable Islet cells, following implantation
into a mammal, may ameliorate the symptoms associated with insulin
deficient diabetes mellitus.
Experimental (pancreatectomized) dogs are anesthetized with
ketamine. While anesthetized, and by means of fluoroscopic
guidance, a titanium Greenfield.RTM. vena cava filter, is
introduced by means of a catheter into a femoral vein by
percutaneous puncture. When the filter is positioned correctly
within the inferior vena cava, as determined by fluoroscopy, the
filter is ejected from the insertion device and anchored to the
wall of the inferior vena cava.
Then, Islet cell containing hollow fibers prepared in accordance
with the optimal conditions found in Example 9 are prepared for
intravenous administration and implantation within the anchored
Greenfield vena cava filter. The hollow fibers are prepared for
injection by encasing them within a fibrin blood clot produced from
blood removed from the host animal. In addition, conventional
radioopaque platinum tags are incorporated into the hollow fibers
to ensure appropriate placement of the fibers and to assist in
retrieval of the fibers after the sacrifice of the animal. Then,
the fibrin clot containing the hollow fibers is administered
through the intravenous catheter upstream of the anchored
Greenfield.RTM. vena cava filter. The fibrin clot containing the
hollow fibers is captured and retained in place within the inferior
vena cava by the filter.
The filter insertion device is removed from the animal, and the
animal is allowed to recover from anesthesia. Then, the insulin and
glucose levels are measured at varying time intervals after
implantation. After a postoperative period ranging from about 2
weeks to 6 about months, the animals are sacrificed and the hollow
fibers retrieved for cell viability analysis. In addition the
levels of insulin protein production in various micro-environments
within the hollow fiber are determined by immunohistochemistry.
Control pancreactomized dogs were treated in exactly the same
manner except that the insulin producing cells were omitted from
the device.
It is anticipated that experimental dogs implanted with the insulin
producing devices exhibit maintainence of euglycemia and have
appropriate plasma insulin levels. It is contemplated, however,
that the control dogs implanted with devices without the insulin
producing cells do not exhibit euglycemia and do not have the
appropriate plasma insulin levels.
Other Embodiments.
The invention may be embodied in other specific forms without
departing from the spirit or essential characteristics thereof. The
present embodiments are therefore to be considered in all respects
as illustrative and not restrictive, the scope of the invention
being indicated by the appended claims rather than by the foregoing
description, and all changes which come within the meaning and
range of equivalency of the claims are therefore intended to be
embraced therein.
Other embodiments of the invention are within the following
claims.
* * * * *